Project Summary/Abstract Platelet activation and aggregation is a major component of thrombosis, a critical underlying cause of myocardial infarction and ischemic stroke. Various anti-platelet drugs are used clinically for prevention of thrombosis. These drugs systemically block platelet activation and aggregation and thus have side effects, such as thrombocytopenia and hemorrhage. Therefore, discovering unique molecular mechanism-mediated anti-platelet and anti-thrombotic targets still has significant priority. We recently demonstrated that thymidine phosphorylase (TYMP) plays a functional role in facilitating multiple agonist-induced platelet activation by finding that TYMP haploinsufficiency significantly inhibits arterial thrombosis without disturbing systemic hemostasis. These exciting findings indicate that modulation of TYMP activity can potentially become a novel and systemically safe anti-platelet therapy. For this, it is first necessary to elucidate the detailed mechanistic pathways of TYMP in platelet activation and thrombosis. Given our current findings, we hypothesize that TYMP plays important functional roles in platelet activation via signaling pathways involving platelet glycoprotein VI (GPVI). We propose to study two specific aims regarding the role of TYMP in platelet aggregation and possible therapeutic avenues for thrombotic diseases. In Aim1, we will conduct studies to determine how TYMP facilitates platelet activation via GPVI signaling using both human and mouse platelets as well as animal models. In Aim 2, we will establish that targeting TYMP in vivo is a safe anti-thrombosis therapy. TYMP inhibitor, tipiracil (TPI), has been approved by the FDA for clinical use, which makes TPI a potential repositioning drug for thrombotic diseases. We will compare the therapeutic effects and the potential side effects of TPI with aspirin and clopidogrel using mouse models. Our early stage findings suggest that TYMP- haploinsufficiency in mice, or pharmacological inhibition of TYMP activity, shows reduction in thrombosis without systemic effect in hemostasis, therefore indicating a therapeutic promise.